Novel RNAi Molecule Delivery Platform Based on Single-siRNA and shRNA Nanocapsules

ABSTRACT

Novel siRNA and shRNA nanocapsules and delivery methods are disclosed herein. These siRNA and shRNA nanocapsules and delivery methods are highly robust and effective. This invention provides a platform for RNAi delivery with low toxicity and long intracellular half-life for practical therapeutic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) to U.S.Application No. 61/612,178 filed Mar. 16, 2012, the disclosure of whichis incorporated by reference in its entirety.

GOVERNMENT RIGHTS

This invention was made with Government support under Grant No.A1069350, awarded by the National Institutes of Health; Grant No.HDTRA1-09-1-0001 awarded by the United States Department of Defense,Defense Threat Reduction Agency. The Government has certain rights inthis invention.

FIELD OF THE INVENTION

This invention relates to the effective delivery of RNA and DNAmolecules into cells by use of novel nanocapsules. Specifically, thisinvention relates to a novel DNA cassette and siRNA nanocapsuletechnology wherein DNA molecules or siRNA are encapsulated within asmall polymer nanocapsule to facilitate delivery to cells.

BACKGROUND

RNA interference is a powerful tool to target and silence specific geneexpression. The term “RNA interference” (RNAi) was coined after thediscovery that injection of double stranded RNA (dsRNA) into C. elegansleads to specific silencing of genes that are highly homologous insequence to the delivered dsRNA (Fire et al., 1998). RNAi is closelylinked to the post-transcriptional gene-silencing (PTGS) mechanism ofco-suppression in plants and quelling in fungi (Catalanotto et al.,2000; Cogoni and Macino, 1999; Dalmay et al., 2000, Ketting andPlasterk, 2000; Mourrain et al., 2000; Smardon et al., 2000).

RNAi was discovered when researchers were attempting to use antisenseRNA to inactivate a C. elegans gene. The researchers found that injectedsense-strand RNA was equally as effective as antisense RNA at inhibitinggene function (Guo et al. (1995) Cell 81: 611-620). Furtherinvestigation revealed that the active agent was modest amounts ofdouble-stranded RNA (dsRNA) that contaminated in vitro RNA preparations.Researchers further determined that exon sequences are required and thatintrons and promoter sequences, while ineffective, did not appear tocompromise RNAi.

RNAi can act systemically. This systemic potency was demonstrated byTimmons and Fire (1998 Nature 395: 854). Timmons and Fire performed asimple experiment that produced an astonishing result. They fed tonematodes bacteria that had been engineered to express double-strandedRNA corresponding to the C. elegans unc-22 gene. The transgenicnematodes developed a phenotype similar to that of unc-22 mutants. Theresults of this and variety of other experiments, in C. elegans andother organisms, indicate that RNAi acts to destabilize cellular RNAafter RNA processing.

Double-stranded RNAs (dsRNAs) can provoke gene silencing in numerous invivo contexts including Drosophila, C. elegans, planaria, hydra,trypanosomes, fungi and plants. Furthermore, short interfering RNA(siRNA), possessing the unique capability to specifically knock down anundesired expression of gene, holds great promises for therapeutics ofdiversified human diseases. In fact, in it was reported in 2009 thatthere were 12 ongoing clinical trials using siRNA to treat diseases.Cheng et al., siRNA Delivery and Targeting, Molecular Pharmaceutics,2009, 6(3):649-650. Of the 12 ongoing trials, 8 trials used naked siRNAfor local treatment of ocular and respiratory diseases. Castanotto, etal., The promises and pitfalls of RNA-interference-based therapeutics,Nature, 2009, 457(7228):426-433. In February 2013, there were 28 siRNAclinical trials reported by the National Institute of Health. SeeNational Institute of Health Clinical Trials website. Many of the 28trials appear to use naked siRNA.

The clinical application of siRNA is constrained by inefficient deliverysystems. Specifically, there is a lack of delivery vehicles that aresafe, stable, and efficient. To date, various delivery systems have beenproposed. Such systems include cationic liposomes, cell-penetratingpeptides (CPPs) and cationic polymers. Tseng et al., Lipid-basedsystemic delivery of siRNA, Advanced Drug Delivery Reviews, 2009,61(9):721-731 and Lewis et al., Systemic siRNA delivery via hydrodynamicintravascular injection, Advanced Drug Delivery Reviews, 2007,59(2-3):115-123.

Cationic liposomes and cationic lipids, such as Lipofectamine® andlipid-like materials, are used widely for in vitro studies with higheffectiveness; however, the toxicity and low efficiency still restraintheir in vivo applications.

For the CPPs-based approaches, RNAi molecules are assembled with CPPs orCPP bioconjugates into complexed particles with significantly improveddelivery efficiency. Crombez et al., A New Potent Secondary AmphipathicCell-penetrating Peptide for siRNA Delivery Into Mammalian Cells,Molecular Therapy, 2009, 17(1):95-103 and Davis et al., Evidence of RNAiin humans from systemically administered siRNA via targetednanoparticles, Nature, 2010, 464(7291):1067-U140. Nevertheless, theformation of such assembled structure was driven by weak noncovalentinteractions and these particles were generally unstable, particularlyagainst serum nucleases which leads to degradation and poor targeting ofthe RNAi.

For the cationic-polymer-based approaches, siRNA are assembled withcationic polymers is mainly through the electrostatic interactions. Theunique proton sponge effect of the cationic polymers provides thecomplexes with improved intracellular delivery efficiency. However,similar to the CPPs-based approach, such assembled systems are unstableand readily dissociate and release their siRNA payload before they reachthe cytoplasm of the target cells.

Accordingly, in spite of such intensive efforts, the design andsynthesis of an effective delivery vehicle for siRNA remainschallenging. Thus, there is an ongoing need to develop novelsiRNA-delivery methods that are highly robust and effective. Success ofthis work will provide a general delivery platform with low toxicity andlong intracellular half-life for practical therapeutic applications.

BRIEF SUMMARY OF THE INVENTION

In certain embodiments, this invention comprises a polymer nanocapsulecomprising a polymer shell and a RNAi molecule. In certain embodiments,the polymer shell comprises one or more positively charged monomersselected from Table 1, one or more crosslinkers selected from Table 2,and one or more neutral monomers selected from Table 3. In certainembodiments, the RNAi molecule is siRNA or an shRNA DNA cassette.

In certain embodiments, one or more crosslinkers comprise a ratio ofdegradable crosslinker to non-degradable crosslinker. In certainembodiments, the ratio of degradable crosslinker to non-degradablecrosslinker is selected from the ratios comprising 1:0, 3:2, 2:3, or1:4. In certain embodiments, all of the crosslinkers are degradablecrosslinkers. In certain embodiments, all the degradable crosslinkersare glycerol 1,3-diglycerolate diacrylate.

In certain embodiments, the one or more positively charged monomers isacryl-spermine. In certain embodiments, the one or more positivelycharged monomers is selected from the group comprising N-(3-Aminopropyl)methacrylamide hydrochloride, Dimethylamino ethyl methacrylate,(3-Acrylamidopropyl)trimethylammonium hydrochloride, and(3-Acrylamidopropyl)trimethylammonium hydrochloride. In certainembodiments, the one or more positively charged monomers is selectedfrom the group comprisingN-(3-((44(3-aminopropyl)amino)butyl)amino)propyl)methacrylamide,N-(3-((4-aminobutyl)amino)propyl)acrylamide,N-(3-((4-aminobutyl)amino)propyl)methacrylamide,N-(2-((2-aminoethyl)(methyl)amino)ethyl)acrylamide,N-(2-((2-aminoethyl)(methyl)amino)ethyl) methacrylamide,N-(piperazin-1-ylmethyl)acrylamide,N-(piperazin-1-ylmethyl)methacrylamide,N-(2-(bis(2-aminoethyl)amino)ethyl)acrylamide, andN-(2-(bis(2-aminoethyl)amino)ethyl)methacrylamide.

In certain embodiments, the polymer nanocapsules comprisesacryl-spermine, tris-acrylamide, and crosslinker selected from Table 2.

In certain embodiments, the one or more positively charged monomers has3 protonable amines. In certain embodiments, the one or more positivelycharged monomers has 2 protonable amines. In certain embodiments, theone or more positively charged monomers has 1 protonable amines.

In certain embodiments, the polymer nanocapsules are approximately 20 nmto 250 nm in diameter.

In certain embodiments, the polymer nanocapsules is conjugated to atargeting agent. In certain embodiments, the targeting agent is selectedfrom the group comprising cyclodextrin, adamantine, CD4, CD8, CD45,aHLA, and transferrin. In certain embodiments, the targeting agentdelivers the polymer nanocapsules to a specific cell type, wherein thecell type is selected from the group comprising immune cells, bloodcells, cardiac cells, lung cells, optic cells, liver cells, kidneycells, brain cells, cells of the central nervous system, cells of theperipheral nervous system, cancer cells, cells infected with viruses,stem cells, skin cells, intestinal cells, and/or auditory cells. Incertain embodiments, the cancer cells are cells selected from the groupcomprising lymphoma cells, solid tumor cells, leukemia cells, bladdercancer cells, breast cancer cells, colon cancer cells, rectal cancercells, endometrial cancer cells, kidney cancer cells, lung cancer cells,melanoma cells, pancreatic cancer cells, prostate cancer cells, andthyroid cancer cells.

In certain embodiments, the polymer nanocapsule further comprises apharmaceutically acceptable carrier.

In certain embodiments, this invention comprises a pharmaceuticalcomposition comprising one or more polymer nanocapsule described above.

In certain embodiments, this invention comprises a method of treating adisease characterized by over expression of a gene with a pharmaceuticalcomposition of a nanocapsules described herein, wherein the siRNA orshRNA knocks down or decreases expression of an over expressed gene,thereby treating the disease.

In certain embodiments, this invention comprises a method of making asiRNA polymer nanocapsules. In certain embodiments, the siRNA isdissolved in RNase-free water. In certain embodiments, one or morepositively charged monomers selected from Table 1 and one or morecrosslinkers selected from Table 2 in deoxygenated and is dissolved indeionized water to create a monomer mixture. In certain embodiments, thedissolved siRNA as described above is combined with the monomer mixture.In certain embodiments, ammonium persulfate andN,N,N′,N′-tetramethylethylenediamine is added to the mixture. In certainembodiments, the mixture is incubated in serum-free medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an illustration of the synthesis and delivery of DNAcassette nanocapsules: 1) self-assembly of hydrophilic monomer A,positively charged monomer B, degradable crosslinker C andnon-degradable crosslinker D around DNA cassette; 2): formation of DNAcassette nanocapsules through in situ polymerization; 3): delivery; IV):release of DNA cassette and expression of siRNA.

FIG. 2 depicts a schematic illustration of the synthesis and delivery ofsingle siRNA nanocapsules. Step I of the schematic depicts a positivelycharged, polymerizable monomer that interacts with the negativelycharged siRNA. Step II of the schematic depicts the polymerization ofpH-degradable crosslinkers and hydrophilic monomers to create a polymernetwork that that wraps each siRNA molecule. Step III of the schematicdepicts crosslinked shell protecting the cored siRNA from hydrolysis.Step IV of the schematic depicts the stability of the polymernanocapsules in serum (pH˜7.4) and their ability to escape from theendosomes upon the degradation of the shell that releases the siRNA tothe cytoplasm.

More specifically, Step I depicts: starting with a positively charged,polymerizable monomer, acryl-spermine (1), electrostatic interactionsenrich 1 around the surface of the negatively charged siRNA. Step IIdepicts: subsequent room-temperature polymerization in an aqueoussolution, which contains the pH-degradable crosslinkers (2) andhydrophilic monomers (3), wraps each siRNA molecule with a thin shell ofpolymer network. Step III depicts: a crosslinked shell well protects thecored siRNA from hydrolysis; while tuning the ratio of 1 and 3 allowsprecise control of the surface charge ensuring the effective cellulartransduction of the polymer nanocapsules. Step IV depicts: (2) is stablein neutral pH but is rapidly degraded in acidic environment, such aslate endosomes with pH˜5.4. It is believed that this unique responsivedesign will provide the polymer nanocapsules with outstanding stabilityin serum (pH˜7.4), while enabling their escape from endosomes upon thedegrading of the shell that releases the siRNA to the cytoplasma.Furthermore, this synthetic approach allows for the immobilization oftargeting components (e.g., antibody) to the polymer nanocapsulessurface, allowing targeted delivery of siRNA.

FIG. 3 depicts images and representations of DNA cassettes andnanocapsules. FIG. 2A depicts a gel electrophoresis image of DNAcassette. Lane a is CCR5 shRNA and lane b is EGFP shRNA. FIG. 2B depictsa TEM image of DNA cassettes (Scale bar=100 nm). FIG. 3C depicts a TEMimage of DNA cassette nanocapsules (molar ratio of DNA to 3 reactantsshown in FIG. 1 and Example 1 and Example 2s A,B,C=1:750:750:30).

FIG. 4 depicts the polymer nanocapsules and their ability to preventdegradation. FIG. 4A depicts a TEM image of siRNA nanocapsules. FIG. 4Bdepicts florescence and optical images of HEK-293 T cells transductedwith FITC-labeled siRNA nanocapsules. FIG. 4C depicts florescence imagesof HEK-293 T cells transducted with si1005 siRNA nanocapsules targetedto CCR5 (right) showing knockdown of CCR5-mCherry fusion protein(control siRNA nanocapsules as control (left)). FIG. 4D depictsCCR5-mCherry knock-down by siRNA nanocapsule (1,3,5,7) and siRNALipofectamine® (Invitrogen®) (2,4,6,8) in the absence or presence ofactive human serum.

FIG. 5 depicts HEK-293T cells transduced with Alexa592-labelled DNAcassette nanocapsules. FIG. 5A depicts an optical image of the cells.FIG. 5B depicts a fluorescent image of the cells. FIG. 5C depicts a flowcytometry graph of the cells. FIG. 5D depicts the knockdown ofCCR5-luciferase by CCR5-shRNA DNA cassette nanocapsules (100 ng DNA per2.5×104 cells in 100 uL). In these experiments, the cells were dosedwith Alexa592-labelled DNA cassette nanocapsules at 100 nM for 4 hours.Then nanocapsules were removed and cells were washed 3 times with PBS.After trypsinization, cells were pictured with Leica Zeiss Axio Observerand also analyzed by a flow cytometer.

FIG. 6 depicts the sensitivity of DNA cassette to Dnase I and siRNA tonulcease and human serum. FIG. 6A depicts the sensitivity of native DNAcassette and DNA nanocapsules to Dnase I. DNA cassette complexed withLipofectamine® and DNA nanocapsules were incubated for 1 hour withoutDnase I and with Dnase I, respectively. Samples were run on 2% agarosegel and imaged with ImageQuant LAS4000. FIG. 6B depicts the sensitivityof siRNA to Nulcease and serum. siRNA complexed with lipofectamine andsiRNA nanocapsules were incubated for 1 hour with nuclease (up) andhuman serum (down), respectively. Then siRNAs were extracted fromlipofectamine and nanocapsules with chloroform/0.1% SDS-0.5 M NaCl.Samples were run on 4% agarose gel and imaged with ImageQuant LAS4000.

FIG. 7 depicts the viability of HEK-293T cells transduced with CCR5 DNAcassette nanocapsules. HEK-293T cells were treated with DNA cassettenanocapsules at 0, 0.1, 0.2 and 0.4 pmol for 4 h at 37° C. in serum-freemedium. Then mediums were changed to DMEM with 10% Bovine Fetal Serum.After 24 h, cell viability was determined through CytoToxGlow kit usinga 96-well plate reader.

FIG. 8 depicts knockdown of CCR5-Luciferase in HEK-293T cells. FIG. 8Adepicts knockdown of CCR5-Luciferase in HEK-293T cells transduced withCCR5 DNA cassette nanocapsules. FIG. 8B depicts knockdown ofCCR5-Luciferase in HEK-293T cells transduced with CCR5 siRNALipofectamine® complex. HEK-293T cells were treated with DNA cassettenanocapsules at 0, 0.1, 0.2 and 0.4 pmol and siRNA Lipofectamine®complex at 0, 50, 100 and 200 pmol for 4 h at 37° C. in serum-freemedium. Then mediums were changed to DMEM with 10% Bovine Fetal Serum.After 48 h, the luciferase activity was determined using a 96-wellsplate reader.

FIG. 9 depicts the degradation rates of CCR5-shRNA DNA cassettenanocpsules. FIG. 9A depicts the degradation rates of CCR5-shRNA DNAcassette nanocapsule prepared with cocktails of degradable andnon-degradable crosslinkers. FIG. 9B depicts the stability of CCR5-shRNADNA cassette nanocapsule monitored by DLS in water. The initial shellthickness of the polymer nanocapsules was estimated from R₀−R*, where R₀is the initial diameter of DNA nanocapsules and R*is the diameter of aDNA molecule (the final diameter after degradation). The degradationdegree was calculated as [R₀−R_(t)]/[R₀−R*]×100%, where R_(t) is thediameter of nanocapsules.

FIG. 10 depicts the down-regulation of CCR5 in HEK293 cells by sh1005DNA cassette nanocapsules with different ratios (5:0; 3:2; 2:3 and 1:4)of degradable crosslinker (Glycerol 1,3-diglycerolate diacrylate, GDGDA)to non-degradable crosslinker (N,N′-methylenesbisacrylamide, BIS). Onday 0 of culture cells were transduced with DNA cassette nanocapsulesfor 4 hours. On day 3, 5 and 9 of culture cells were labeled withanti-CCR5 and analyzed by flow cytometry.

FIG. 11 depicts the ability of nanocapsules prepared with positivelycharged monomers #1-#14 from Table 1 to knockdown luciferase geneexpression in luciferase-expressing CWR cells. As can be seen from thefigure, the best knockdown of luciferase expression was achieved withnanocapsules prepared with positively charged monomers #1, #2, #7 and#8.

FIG. 12 depicts the ability of nanocapsules prepared with crosslinkers#1-#5 from Table 2 to knockdown luciferase gene expression inluciferase-expressing CWR cells. As can be seen from the figure, thebest knockdown of luciferase expression was achieved with nanocapsulesprepared with crosslinker #1 (i.e., 2-hydroxyethyl methacrylate).

FIG. 13 depicts the ability of neutral monomers to affect the size ofthe siRNA nanocapsules. As can be seen in the figure,N-(1,3-dihydroxy-2-(hydroxymethyl) propan-2-yl) acrylamide produced thesmallest nanocapsules (less than 25 nm) and acrylamide produced thelargest nanocapsules (greater than 200 nm).

FIG. 14 depicts the knockdown of luciferase gene expression by usingluciferase siRNA nanocapsules. FIG. 14A depicts the knockdown ofluciferase gene expression in CWR cells using luciferase siRNAnanocapsules and control siRNA nanocapsules. FIG. 14B depicts the cellviability after treatment of siRNA nanocapsules. CWR cells were treatedwith siRNA nanocapsules at 0, 20, 50, 100, 200 and 300 nM for 4 h at 37°C. in serum-free medium. Then mediums were changed to DMEM with 10%Bovine Fetal Serum. After 48 h, the luciferase activity was determinedusing a 96-wells plate reader.

FIG. 15 depicts the pH titration curve of nanocapsules and confirms thatone of the polymer nanocapsules has about 60% of buffer capacity ofPEI25K between pH 7.3 and 5.5. The buffering capacity of the nanocapsulewas measured by acid-base titration. The nanocapsule solutions (8.3 mMin terms of total molar concentration of ioniziable amine groups), whichwere initially adjusted to pH 10, were titrated with 0.01M HCl. The pHprofiles were recorded at room temperature.

FIG. 16 depicts the TEM picture of BSA control nanoparticle. It issynthesized using the same formulation as that of siRNA nanocapsules anddoes not show the dark cores in the image. BSA was dissolved in 20 uLRnase-free water at 20 uM. Then a specific amount of acryl-spermine,tris-acrylamide and glycerol dimethacrylate (molar ratio=5:5:1)dissolved in 0.5 mL deoxygenated and deionized water was mixed with BSAin the microcentrifugetube (final molar ratio of BSA toacryl-spermine=1:220). Radical polymerization from the surface of BSAwas initiated by adding 2 uL of 1% ammonium persulfate solution and 1 μLof 5% N,N,N′,N′-tetramethylethylenediamine (final molar ratio of BSA toacryl-spermine=1:240). The reaction was allowed to proceed for 60 min ina nitrogen atmosphere.

FIG. 17 depicts the surface charge of one of the siRNA nanocapsulesdetermined by dynamic light scattering.

FIG. 18 depicts the size and size distribution of one of the siRNAnanocapsules determined by dynamic light scattering. The averagediameter of 24.6 nm obtained by light scattering is consistent with theaverage diameter of 20 nm obtained by TEM.

FIG. 19 depicts the knockdown of luciferase by one of the siRNAnanocapsules in the presence and absence of serum.

FIG. 20 depicts the degradation profile of siRNA nanocapsules under pH5.4 or pH 7.4.

FIG. 21 depicts the absorption change of siRNA after self-assemblingwith monomers. The absorption of siRNA (top) decreased after formationof siRNA-monomers complex (bottom).

FIG. 22 depicts the targeting delivery of siRNA to 293 cells expressingCD4 receptors.

FIG. 22A shows the flow cytometry graph of 293 cells expressing CD4receptors after incubation with FITC-labeled siRNA nanocapsulesconjugated with anti-CD4 antibody for 4 hours. FIG. 22B shows the flowcytometry graph of regular 293 cells without CD4 receptors afterincubation with FITC-labeled siRNA nanocapsules conjugated with anti-CD4antibody for 4 hours. 100 uL of anti-CD4 antibody dissolved in pH=8.7HEPES buffer at 1 mg/mL reacted with 10 uL of 2-Iminothiolane (Trout'sreagent) at 1 mg/mL for 30 minutes. The modified anti-CD4 antibody willbe then incubated with 100 uL of FITC-labeled siRNA nanocapsules at 10uM overnight.

DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below.

As used herein, the terms “target” refers to a section of a DNA or RNAstrand of a double-stranded DNA or an RNA that is complementary to asection of a DNA or RNA strand, including all transcribed regions, thatserves as a matrix for transcription. A target gene, usually the sensestrand, is a gene whose expression is to be selectively inhibited orsilenced through RNA interference.

As used herein, the term “polymer nanocapsules” refers to a compositioncomprising a “polymer shell” and a “RNAi molecule.”

As used herein, the term “polymer shell” refers to the polymer portionof the RNAi polymer nanocapsules comprising one or more positivelycharged polymer monomers, one or more crosslinkers, and optionally oneor more neutral polymer monomers. Examples of positively chargedmonomers, crosslinkers, and neutral monomers are provided in Table 1,Table 2, and Table 3.

As used herein, the term “RNAi molecule” refers to a RNA or DNA moleculethat plays a role in RNA interference. Specifically, an RNAi moleculerefers to a shRNA, siRNA, or dsRNA as disclosed herein. A small hairpinRNA (shRNA) is a RNA sequence that forms a tight hairpin turn that canbe used to silence gene expression by RNA interference. shRNAs can bedelivered to target cells using DNA plasmids, viral vectors, orbacterial vectors. As used herein, shRNA can be delivered to cells usingDNA cassettes. Double-stranded RNAs (dsRNA) comprise a broad group ofviruses. Small interfering RNA (siRNA) is a class of double stranded RNAmolecules. siRNAs are short, generally around 20-25 base pairs inlength. siRNA can be used to silence gene expression by RNAinterference. Furthermore, siRNA can also act in RNAi-related pathwayssuch as viral and retroviral infection.

As used herein, the terms “degradable polymer” and “nondegradablepolymer” refer to the ability of the polymers described herein todegrade into smaller fragments. In certain embodiments of thisinvention, degradable polymers can break down at physiological pH. Incertain embodiments, degradable polymers can break down at approximatelypH 7.4. In certain embodiments, a mixture of degradable andnondegradable polymers can yield a degradable polymer mixture. Incertain embodiments, a mixture of degradable and nondegradable polymerscan break down at physiological pH. In certain embodiments, a mixture ofdegradable and nondegradable polymers can break down at approximately pH7.4.

As used herein, the terms “mutant gene” and “target mutant gene” referto a gene comprising at least one point mutation relative to thecorresponding normal, non-mutated cellular gene (referred to herein asthe “corresponding wild-type gene”). The terms mutant gene and targetmutant gene specifically encompass any variant of a normal cellular geneor gene fragment whose expression is associated with a disease ordisorder (e.g., an oncogene).

As used herein, the term “conjugate” or “conjugate agent” or“surface-conjugated targeting agent” or “polymer nanocapsule conjugates”refers to any moiety, such as a protein or effective portion thereof,that is conjugated to the polymer nanocapsules and provides specifictargeting of the polymer nanocapsules to the surface of a specific celltype thereby providing directed delivery of the siRNA or shRNA to aspecific cell type. For example, the conjugate agent can binding to acell-specific cell surface receptor, thereby bringing the polymernanocapsules into immediate proximity to the target cell. In certainembodiments, the conjugates used to achieve specific targeting of thepolymer nanocapsules include CD4, CD8, CD45, CD133, aHLA, andtransferrin. In other embodiments, the conjugates can be cell-specificantibodies or fragments thereof. Additional examples of conjugates usedto target specific cell types are described below in the DetailedDescription.

The term “complementary RNA strand” (also referred to herein as the“antisense strand”) refers to the strand of a dsRNA which iscomplementary to an mRNA transcript that is formed during expression ofthe target gene, or its processing products. As used herein, the term“complementary nucleotide sequence” refers to the region on thecomplementary RNA strand that is complementary to a region of an mRNAtranscript of the target mutant gene (i.e., “the correspondingnucleotide sequence” of the target gene). “dsRNA” refers to aribonucleic acid molecule having a duplex structure comprising twocomplementary and anti-parallel nucleic acid strands. Not allnucleotides of a dsRNA must exhibit Watson-Crick base pairs. The maximumnumber of base pairs is the number of nucleotides in the shortest strandof the dsRNA. The RNA strands may have the same or a different number ofnucleotides. The complementary nucleotide region of a complementary RNAstrand is less than 25, preferably 19 to 24, more preferably 20 to 24,even more preferably 21 to 23, and most preferably 22 or 23 nucleotidesin length. The complementary RNA strand is less than 30, preferablyfewer than 25, more preferably 21 to 24, and most preferably 23nucleotides in length. dsRNAs comprising a complementary or antisensestrand of this length (known as “short interfering RNA” or “siRNA”) areparticularly efficient in inhibiting the expression of the target mutantgene. “Introducing into” means uptake or absorption in the cell, as isunderstood by those skilled in the art. Absorption or uptake of dsRNAcan occur through cellular processes, or by auxiliary agents or devices.For example, for in vivo delivery, dsRNA can be injected into a tissuesite or administered systemically. In vitro delivery includes methodsknown in the art such as electroporation and lipofection.

As used herein, “selective inhibition of expression” means that a dsRNAhas a greater inhibitory effect on the expression of a target mutantgene than on the corresponding wild-type gene. Preferably, theexpression level of the target mutant gene is less than 98%, less than95%, less than 90%, less than 80%, less than 70%, less than 60%, lessthan 50%, less than 40%, less than 30%, less than 20%, or less than 10%of the expression level of the corresponding wild-type gene.

As used herein and as known in the art, the term “identity” is therelationship between two or more polynucleotide sequences, as determinedby comparing the sequences. Identity also means the degree of sequencerelatedness between polynucleotide sequences, as determined by the matchbetween strings of such sequences. Identity can be readily calculated(see, e.g., Computation Molecular Biology, Lesk, A. M., eds., OxfordUniversity Press, New York (1998), and Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York (1993),both of which are incorporated by reference herein). While there exist anumber of methods to measure identity between two polynucleotidesequences, the term is well known to skilled artisans (see, e.g.,Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press(1987); and Sequence Analysis Primer, Gribskov., M. and Devereux, J.,eds., M. Stockton Press, New York (1991)). Methods commonly employed todetermine identity between sequences include, for example, thosedisclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math. (1988)48:1073. “Substantially identical,” as used herein, means there is avery high degree of homology (preferably 100% sequence identity) betweenthe sense strand of the dsRNA and the corresponding part of the targetgene. However, dsRNA having greater than 90% or 95% sequence identitymay be used in the present invention, and thus sequence variations thatmight be expected due to genetic mutation, strain polymorphism, orevolutionary divergence can be tolerated. Although 100% identity ispreferred, the dsRNA may contain single or multiple base-pair randommismatches between the RNA and the target gene.

As used herein, the term “treatment” refers to the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disorder, e.g., a disease or condition, asymptom of disease, or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve, or affect the disease, the symptoms of disease, or thepredisposition toward disease.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

DETAILED DESCRIPTION OF THE INVENTION Introduction

RNA interference is a powerful tool to silence specific gene expressionand a variety of RNAi-based therapies are being considered for humandiseases. However, the major impediment to the effective use of RNAi forapplications in humans is its effective delivery into cells. Forexample, transduction of short hairpin shRNA into hematopoietic stemcells by lentiviral vector has shown to allow endogenous synthesis ofsiRNA and is able to provide sustainable gene silencing. However, thetoxic myeloablative regimens used in the transplant procedure and thelimited engraftment of gene-modified cells currently constrains wideapplication.

A variety of non-viral siRNA delivery systems have been proposed,including cationic liposomes, cell-penetrating peptides (CPPs) andcationic polymers. Cationic lipids, such as Lipofectamine® andlipid-like materials, are widely used for in vitro studies and haveshown potential for in vivo gene silencing especially in the liver. Asan example of the CPPs-based approach, siRNA can be assembled with CPPsor CPP bioconjugates into complexed particles to generate significantlyimproved delivery efficiency. Nevertheless, the formation of suchstructures is driven by weak non-covalent interactions and theseparticles were generally unstable, particularly, against serumnucleases.

For the cationic-polymer-based approach, siRNA can be assembled withcationic polymers mainly through electrostatic interactions. Forexample, cationic polymer-based nanoparticles with transferrin as atargeting agent have been shown to deliver siRNA molecules to tumors inhumans and reduce the expression of the ribonucleotide reductasesub-unit RRM2, an anti-cancer target. However, similar to the CPP-basedapproach, such systems are based upon non-covalent electrostaticself-assembly, which have competition from electrolytes and polyionswithin the bloodstream. Therefore, in spite of intensive efforts, thedesign and synthesis of an effective delivery vehicle for siRNA remainsa challenge.

Despite the challenges described above, siRNA has become one of the mostpromising and specific drug candidates with broad potential for thetreatment of diverse human diseases attributed to their unique abilityto regulate specific genes and control the expression of correspondingproteins. Current commercial products for in vitro siRNA deliveryinclude lipofection and nucleofection. Although these two methods arewidly applicable in many cell lines, the delivery efficiency and thetoxicity in primary cells remains a formidable challenge.

For in vivo delivery of siRNA, siRNA-mediated gene silencing in mammalsthrough systemic administration was achieved using naked siRNA. Howeverthis administration is not compatible with and has many limitations foruse in human. Several recent studies have shown improved siRNA deliveryin mouse models and nonhuman primates using positively charged peptideor proteins like arginine peptide, CpG oligonucleotide and protamine.Crombez et al., A New Potent Secondary Amphipathic Cell-penetratingPeptide for siRNA Delivery Into Mammalian Cells, Molecular Therapy,2009, 17(1):95-103. Another recent study demonstrates the presence of anRNAi mechanism in humans through systemic administration of siRNA ananoparticles targeted to melanoma cells. Davis et al., Evidence of RNAiin humans from systemically administered siRNA via targetednanoparticles, Nature, 2010, 464(7291):1067-1070. Nevertheless, highlyeffective delivery to targeting sites still persists as a majorobstacle. In addition, potential adverse immune responses caused by thesiRNA remains to be solved.

As described herein, highly stabilized RNAi molecule polymernanocapsules (“polymer nanocapsules”) were fabricated through an in situpolymerization technique, which introduced a protective crosslinkeddegradable polymer shell covering the surface of the RNAi molecules withdesigned charges and properties. This crosslinked polymer shell providesprotection to the RNAi molecules from RNase degradation, temperaturedissociation and serum inactivation. Furthermore, degradability of thepolymer network assigns a control-releasing property to RNAi molecules,which results in a less intracellular immune response. By adjusting thecharge of the polymer nanocapsules, delivery efficiency of RNAimolecules into human cells was achieved. Furthermore, efficient RNAimolecule delivery into a broad variety of cells including 293T, Hela,CEM, and PBMCs was achieved with highly positive-charged nanocapsules.

Furthermore, surface-conjugated targeting agents on optimizednanocapsules provided successful targeted delivery of RNAi moleculesinto cells of interest, such as T-cells. The diversified andcontrollable nontargeting and targeting abilities provided to the RNAimolecule delivery will have important implications for many in vitrotests and clinical applications using RNAi molecules to knock down anydesired gene expressions.

Polymer Nanocapsules

This invention provides a novel strategy through self-assembly and insitu polymerization technology to imprint RNAi molecules intocrosslinked polymer nanocapsules. In certain embodiments, the polymernanocapsules are approximately 20-100 nm in diameter. The smalldiameters of the polymer nanocapsules maximize the protection of theRNAi molecules from external RNase attack and serum neutralization.

In specific embodiments, the polymer nanocapsules are 10 nm-20 nm, 20-25nm, 25 nm-30 nm, 30 nm-35 nm, 35 nm-40 nm, 40 nm-45 nm, 45 nm-50 nm, 50nm-55 nm, 55 nm-60 nm, 60 nm-65 nm, 70-75 nm, 75 nm-80 nm, 80 nm-85 nm,85 nm-90 nm, 90 nm-95 nm, 95 nm-100 nm, or 100 nm-110 nm. In specificembodiments, the polymer nanocapsules are approximately 10 nm, 11 nm, 12nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42nm, 43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62nm, 63 nm, 64 nm, 65 nm, 66 nm, 67 nm, 68 nm, 69 nm, 70 nm, 71 nm, 72nm, 73 nm, 74 nm, 75 nm, 76 nm, 77 nm, 78 nm, 79 nm, 80 nm, 81 nm, 82nm, 83 nm, 84 nm, 85 nm, 86 nm, 87 nm, 88 nm, 89 nm, 90 nm, 91 nm, 92nm, 93 nm, 94 nm, 95 nm, 96 nm, 97 nm, 98 nm, 99 nm, 100 nm, 101 nm, 102nm, 103 nm, 104 nm, 105 nm, 106 nm, 107 nm, 108 nm, 109 nm, 110 nm, 111nm, 112 nm, 113 nm, 114 nm, 115 nm, 116 nm, 117 nm, 118 nm, 119 nm, or120 nm in diameter. In specific embodiments the polymer nanocapsules are120 nm-130 nm, 130 nm-140 nm, 140 nm-150 nm, 150 nm-160 nm, 160 nm-170nm, 170 nm-180 nm, 180 nm-190 nm, 190 nm-200 nm, 200 nm-210 nm, 220nm-230 nm, 230 nm-240 nm, 240 nm-250 nm, or larger than 250 nm indiameter.

In certain embodiments, additional advantages of the polymernanocapsules disclosed herein include nontargeting and targetingability, higher efficiency, and lower adverse immune response. Forexample, the higher efficiency may result from increased uptake and moredirected delivery.

Furthermore highly stabilized RNAi molecules inside the protectivenanocapsule is able to be fully released once the nanostructured polymershell is degraded in endosomes and lysosomes. In certain embodiments, anontargeting polymer encapsulated RNAi molecules can be transduced intoprimary cells such as PBMCs in vitro with superior efficiency andnoncytoxicity compared to the low efficiency and high toxicity resultingfrom liposome transduction.

Importantly, by choosing and designing appropriate polymer charge, themethod of RNAi molecule delivery to specific purposes (such as targetingby conjugating moieties to the polymer nanocapsules as described herein)can be modulated.

In certain embodiments, the ratio of degradable crosslinker tonon-degradable crosslinker is 1:1, 1:2, 2:1, 1:3, 2:3, 3:1, 3:2, 4:1,1:4, 4:3, 3:4, 5:1, 1:5, 2:5, 5:2, 5:3, 3:5, 4:5, 5:4, 6:1, 1:6, 1:7,7:1, 2:7, 7:2, 3:7, 7:3, 4:7, 7:4, 5:7, 7:5, 6:7, 7:6, 8:1, 1:8, 3:8,8:3, 5:8, 8:5, 7:8, 8:7, 9:1, 1:9, 2:9, 9:2, 4:9, 9:4, 5:9, 9:5, 7:9,9:7, 8:9, 9:8, 10:1, 1:10, 3:10, 10:3, 7:10, 10:7, 9:10, 10:9 or anyother ratio that one of skill in the art would know to use.

In certain embodiments, the degradable crosslinkers are one or more ofcrosslinker 1, 2, 3, 4, or 6 in Table 2. In certain embodiments, thenon-degradable crosslinkers are crosslinker 5 in Table 2.

In certain embodiments, the polymer nanocapsules are designed to degradein 1 hour, or 2 hours, or 3 hours, or 4 hours, or 5 hours, or 6 hours,or 7 hours, or 8 hours, or 9 hours, or 10 hours, or 11 hours, or 12hours, or 13 hours, or 14 hours, or 15 hours, or 16 hours, or 17 hours,or 18 hours, or 19 hours, or 20 hours, or 21 hours, or 22 hours, or 23hours, or 1 day, or 2 days, or 3 days, or 4 days or 5 days, or 6 days,or 1 week, or 2 weeks, or 3 weeks, or 1 month or any combinationthereof. In certain embodiments, the polymer nanocapsules are designedto degrade at any of the above rates at a physiological pH. In specificembodiments, the polymer nanocapsules are designed to degrade at any ofthe rates above post-administration to a subject in need thereof.

In specific embodiments, RNAi molecules can be effectively delivered tospecific sites in vivo. In certain embodiments, a targeting agent (i.e.,a conjugate or conjugate agent) is conjugated to the polymernanocapsule. In certain embodiments the conjugation prevents thedissociation of the targeting agent from the polymer particle. Inspecific embodiments, cyclodextrin and adamantane can be used fortargeting agent conjugation.

In one embodiment, the invention is practiced using nontargeted andtargeted polymer nanocapsules RNAi molecule delivery with highefficiency and low toxicity for in vitro testing and in vivo targetingto specific tissues and organs via intravenous injection.

The enhanced stability of RNAi molecules attributed to the cross-linkedpolymer also ensures its long-lasting circulation in body before itreaches the targeting sites. Overall, the novel RNAi molecule deliverytechnology described herein has a notable efficiency, augmentedstability, and minimal toxicity both in vitro and predicted in vivo.

Monomers and Cross Linkers

Different monomers and crosslinkers can be used to encapsulate the RNAimolecules by in situ polymerization.

In certain embodiments, the positive monomer have the structure:

-   -   wherein    -   R²⁰ is unsubstituted C₁-C₆ alkyl    -   R²¹ is selected from the group consisting of:

-   -   wherein m is an integer from 1 to 5;    -   R²² is H or unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substituted        with NR²⁵R²⁶, wherein        -   R²⁵ and R²⁶ are independently selected from H or            unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substituted with            amino or C₁-C₆ alkyl substituted with NR²⁷R²⁸, wherein        -   R²⁷ and R²⁸ are independently selected from H or            unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substituted with            amino    -   R²³ is H or unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substituted        with amino or C₁-C₆ alkyl substituted with NR²⁹R³⁰, wherein        -   R²⁹ and R³⁰ are independently selected from H or            unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substituted with            amino or C₁-C₆ alkyl substituted with NR³¹R³², wherein        -   R³¹ and R³² are independently selected from H or            unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substituted with            amino    -   and R²² and R²³ are optionally combined to form a 5-7 membered        heterocycloalkyl ring; and    -   R²⁴ is a lone pair of electrons or unsubstituted C₁-C₆ alkyl.

In certain embodiments, the crosslinkers have the structure:

-   -   wherein R¹ is unsubstituted C₁-C₆ alkyl,    -   R⁴ is unsubstituted C₁-C₆ alkyl,    -   and A is selected from the group consisting of:

-   -   wherein R¹⁰ is unsubstituted C₁-C₆ alkylene;    -   an amino acid;    -   a peptide consisting of from 2 to 10 amino acids;

-   -   wherein R¹¹ is unsubstituted C₁-C₆ alkylene and R¹² is        unsubstituted C₁-C₆ alkylene;

-   -   wherein n is from 1 to 10; and

-   -   wherein R¹³ is unsubstituted C₁-C₆ alkylene and R¹⁴ is        unsubstituted C₁-C₆ alkylene.

In certain embodiments, the neutral monomers have the structure:

wherein R1 is unsubstituted C1-C4 alkyl and R2 is amino or aminosubstituted with hydroxy substituted alkyl or OR³, wherein R3 is hydroxyalkyl.

Non-limiting examples of monomers are presented in Tables 1 and 3.Non-limiting examples of crosslinkers can be found in Table 2. Targetingagents, such as antibodies, peptides, or growth factors can becovalently or noncovalently conjugated with the polymer as describedbelow.

TABLE 1 Non-Limiting Examples of Positively Charged Monomers For RNAiMolecule Polymer Nanocapsules (* monomers are not commercially availableand were specifically synthesized for the manufacture of thesenanocaspules) Number of Protonable # Name Structure amines 1N-(3-((4-((3- aminopropyl)amino)butyl)amino) propyl)acrylamide*

3 2 N-(3-((4-((3- aminopropyl)amino)butyl)amino) propyl)methacrylamide*

3 3 N-(3-((4- aminobutyl)amino)propyl)acrylamide*

2 4 N-(3-((4- aminobutyl)amino)propyl) methacrylamide*

2 5 N-(2-((2- aminoethyl)(methyl)amino)ethyl) acrylamide*

1 6 N-(2-((2- aminoethyl)(methyl)amino)ethyl) methacrylamide*

1 7 N-(piperazin-1- ylmethyl)acrylamide*

2 8 N-(piperazin-1- ylmethyl)methacrylamide*

2 9 N-(2-(bis(2- aminoethyl)amino)ethyl)acrylamide*

3 10 N-(2-(bis(2- minoethyl)amino)ethyl)methacrylamide*

3 11 N-(3-Aminopropyl) methacrylamide hydrochloride

1 12 Dimethylamino ethyl methacrylate

1 13 (3-Acrylamidopropyl) trimethylammonium hydrochloride

1 14 2-aminoethyl methacrylate

1

TABLE 2 Non-Limiting Examples of Crosslinkers for RNAi molecule PolymerNanocapsules. # Name Structure 1 1,3-glycerol dimethacrylate

2 Glycerol 1,3-diglycerolate diacrylate

3 N,N′-bis(acryloyl)cystamine

4 bis[2-(methacryloyloxy)ethyl]phosphate

5 N,N′-Methylenebisacrylamide

6 bisacryloylated polypeptide

TABLE 3 Non-Limiting Examples of Neutral Monomers for RNAi MoleculePolymer Nanocapsules # Name Structure 1 N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2- yl)acrylamide

2 acrylamide

3 N-(hydroxymethyl)acrylamide

4 2-hydroxyethyl acrylate

5 2-hydroxyethyl methacrylate

Polymer Nanocapsule Conjugates

In certain embodiments, targeted delivery of RNAi molecules into cellsis achieved using surface-conjugated targeting agents on optimizednanocapsules. Of particular interest, the polymer nanocapsule conjugatescan be used to target immune, pulmonary, lung, optic, liver, kidney,brain, central nervous system, peripheral nervous system, cardiac,cancer, proliferative, virally or retrovirally infected, stem, skin,intestinal, and/or auditory cells.

In certain embodiments, the conjugates used to achieve specifictargeting of the polymer nanocapsules include CD4, CD8, CD45, CD133,aHLA, and transferrin. In certain embodiments, the conjugates used toachieve specific targeting of the polymer nanocapsules include any oneor more of the cluster of differentiation or cluster of designation (CD)markers. For example, the CD markers include CDX wherein X can be anyone of 1-340. As described herein, the term “CD1”, for example, meansall CD1 variants and subtypes. This applies to all CD markers describedherein.

In certain embodiments, the conjugates used to achieve specifictargeting of the polymer nanocapsules include any one or more of AFP,beta-Catenin, BMI-1, BMP-4, c-kit, CXCL12, SDF-1, CXCR4, decorin,E-Cadherin, Cadherin 1, EGFR, ErbB1, Endoglin, EpCAM, TROP-1, Fc epsilonRI A, FCER1A, L1CAM, LMO2, Nodal, Notch-1, PDGFRB, Podoplanin, PTEN,Sonic Hedgehog, STAT3, Syndecan-1, Tranferrin Receptor, and Vimentin.

In certain embodiments, the conjugates used to achieve specifictargeting of the polymer nanocapsules include any one or more of ALK,AFP, B2M, Beta-hCG, BCR-ABL, BRAF, CA15-3, CA19-9, CA-125, Calcitonin,CEA (Carcinoembryonic antigen), CD20, Chromagranin A, Cytokeratin orfragments thereof, EGFR, Estrogen Receptor, Progesterone Receptor,Fibrin, Fibrinogen, HE4, HER2/neu, IgG variants, KIT, lactatedehydrogenase, Nuclear matrix protein 22, PSA, thyroglobulin, uPA,PAI-1, and Oval.

Any marker described herein or known to one of skill in the art can beused alone, or in combination with one or more additional markers, toachieve the desired targeting of specific cells.

All other cell and/or tissue specific markers known to one of skill inthe art here incorporated by reference.

Methods of Treating Diseases Caused by Expression of a Target Gene

In one embodiment, the invention relates to a method for treating asubject having a disease or at risk of developing a disease caused bythe expression of a target mutant gene. In certain embodiments, thepolymer nanocapsules described herein can act as novel therapeuticagents for controlling one or more of cellular proliferative and/ordifferentiative disorders. In certain embodiments, the polymernanocapsules can act as novel therapeutic agents for controlling one ormore immune or immunodeficiency disorders. In certain embodiments, thepolymer nanocapsules can act as novel therapeutic agents for preventingviral replication or viral infection. In certain embodiments, thepolymer nanocapsules can act as novel therapeutic agents for controllingone or more neurological or neurodegenerative disorders. In certainembodiments, the polymer nanocapsules can act as novel therapeuticagents for treating or preventing cancer.

In certain embodiments, the polymer nanocapsules can act as noveltherapeutic agents for treating advanced cancers, pachyonychiacongenital, age-related macular degeneration, choroidalneovascularization, metastatic melanoma, metastatic melanoma without CNSmetastases, chronic myeloid leukemia, solid tumors, advanced solidtumors, optic atrophy, non-arteric anterior ischemic optic neuropathy,pancreatic cancer, pancreatic ductal adenocarcinoma, diavetic macularedema, hypercholesterolemia, colorectal cancer with hepatic metastases,pancreatic cancer with hepatic metastases, gastric cancer with hepaticmetastases, breast cancer with hepatic metastases ovarian cancer withhepatic metastases, preeclampsia, neuroblastoma, ocular hypertension,open angle glaucoma, glaucoma, ocular pain, dry eye syndrome, kidneyinjury, acute renal failure, delayed graft function, complications ofkidney transplant, TBX3 overexpression, and diabetic retinopathy.

In specific embodiments, the polymer nanocapsules can act as noveltherapeutic agents for treating viral infections. In specificembodiments, the polymer nanocapsules can act as novel therapeuticagents for treating retroviral viral infections. In specificembodiments, the polymer nanocapsules can act as novel therapeuticagents for treating HIV or AIDS infections. In specific embodiments, thepolymer nanocapsules can act as novel therapeutic agents for suppressingretroviral viral infections. In specific embodiments, the polymernanocapsules can act as novel therapeutic agents for blocking,preventing, or downregulating retrovirus or virus replication.

In certain embodiments, the polymer nanocapsules described herein can beadministered by intravitreal injection, intravenously, by injection intothe callus on the bottom of one foot, by oral administration,subcutaneously, and by any other mode of pharmaceutical administrationknown to one of skill in the art.

In certain embodiments, the method comprises administering apharmaceutical composition of the invention to the subject, such thatexpression of the target mutant gene is silenced or down regulated. Incertain embodiments the subject is a mammal. In certain embodiments themammal is a human. Because of their high specificity, the polymernanocapsules of the present invention specifically target the mutantgenes of diseased cells and tissues, as described herein.

In the prevention of disease, the target gene may be one which isrequired for initiation or maintenance of the disease, or which has beenidentified as being associated with a higher risk of contracting thedisease. In the treatment of disease, the polymer nanocapsules can bebrought into contact with the cells or tissue exhibiting the disease. Asone non-limiting example, polymer nanocapsules are substantiallyidentical to all or part of a mutated gene associated with cancer, orone expressed at high levels in tumor cells may be brought into contactwith or introduced into a cancerous cell or tumor gene. As anothernon-limiting example, polymer nanocapsules are substantially identicalto all or part of a mutated gene associated with a viral or retroviraldisease. Specifically, a non-limiting example of a retroviral diseasethat can be treated with the polymer nanocapsules described herein isHIV (see FIGS. 3, 4, and 6-10).

Non-limiting examples of cellular proliferative and/or differentiativedisorders include cancer, e.g., carcinoma, sarcoma, metastatic disordersor hematopoietic neoplastic disorders, e.g., leukemias. A metastatictumor can arise from a multitude of primary tumor types, including butnot limited to those of prostate, colon, lung, breast and liver origin.As used herein, the terms “cancer,” “hyperproliferative,” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state of condition characterized by rapidlyproliferating cell growth. These terms are meant to include all types ofcancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. Proliferative disordersalso include hematopoietic neoplastic disorders, including diseasesinvolving hyperplastic/neoplatic cells of hematopoietic origin, e.g.,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof.

Mutations in cellular genes that directly or indirectly control cellgrowth and differentiation are considered to be the main cause ofcancer. There are approximately thirty families of genes, calledoncogenes, which are implicated in human tumor formation. Members of onesuch family, the RAS gene family, are carried in a broad range ofeukaryotes and are frequently found to be mutated in human tumors.Polymer nanocapsules of this invention can be used to target suchoncogenes to knock down or prevent their expression.

In addition to oncogenes, the methods and compositions of the inventioncan be applied to other disease-related target genes having a pointmutation. Gene mutations have been reported in more than 1000 differenthuman genes. Data on these mutations and their associated phenotypeshave been collated and are available online through two major databases:Online Mendelian Inheritance in Man in Baltimore and the Human GeneMutation Database in Cardiff. For example, there is a high frequency ofCG to TG or CA mutations in the human genome due to deamination of 5′methyl-cytosine. Short deletions or insertions of less than 20nucleotides are also very common mutations in humans. See, e.g.,Antonarakis, S. E., Eur. Pediatr. (2000) 159(3):5173-8.

Furthermore, Sachidanandam et al. describes a map of human genomesequence variation containing 1.42 million single nucleotidepolymorphisms, which is useful for identifying biomedically importantgenes for diagnosis and therapy (Sachidanandam, R., et al., Nature(2001) 409(6822):821-2 and Nature (2001) 409(6822):822-3). The mapintegrates all publicly available SNPs with described genes and othergenomic features. An estimated 60,000 SNPs fall within exon (coding anduntranslated regions), and 85% of exons are within 5 kb of the nearestSNP. Clifford et al. provides expression-based genetic/physical maps ofsingle-nucleotide polymorphisms identified by the cancer genome anatomyproject (Clifford, R., et al., Genome Res (2000) 10(8):1259-65). Inaddition to SNP maps, Sachidanandam et al. provide maps containing SNPsin genes expressed in breast, colon, kidney, liver, lung, or prostatetissue.

Accordingly, RNAi molecule polymer nanocapsules of this invention can beused to target such mutant genes to knock down or prevent theirexpression

Methods of Inhibiting Expression of a Mutant Gene

In yet another aspect, the invention relates to a method for inhibitingthe expression of a mutant gene in subject. The method comprisesadministering a composition of the invention to the subject such thatexpression of the mutant gene is silenced as compared to thecorresponding wild-type gene. In certain embodiments the subject is amammal. In certain embodiments the mammal is a human.

Because of their high specificity, the siRNA nanocapsules of the presentinvention specifically target RNAs (primary or processed) of targetmutant genes, and at surprisingly low dosages. Compositions and methodsfor inhibiting the expression of a target gene using polymernanocapsules can be performed as described herein.

In one embodiment, the invention comprises administering a compositioncomprising polymer nanocapsules, wherein the polymer nanocapsulescomprise a nucleotide sequence which is substantially complementary toan RNA transcript of the target mutant gene and partially complementaryto the corresponding wild-type gene. When the subject to be treated is amammal, such as a human, the composition may be administered by anymeans known in the art including, but not limited to oral or parenteralroutes, including intravenous, intramuscular, intraperitoneal,subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical(including buccal and sublingual) administration. In preferredembodiments, the compositions are administered by intravenous orintraparenteral infusion or injection.

The methods for inhibition the expression of a target gene can beapplied to any mutant gene one wishes to silence, thereby selectivelyinhibiting its expression. Non-limiting examples of human genes whichcan be targeted for silencing include oncogenes cytokinin gene, idiotypeprotein genes, prion genes, genes that expresses molecules that induceangiogenesis, genes that encode adhesion molecules, genes that encodecell surface receptors, genes of proteins that are involved inmetastasizing and/or invasive processes, genes of proteases as well asof molecules that regulate apoptosis and the cell cycle, genes thatexpress the EGF receptor, genes that encode the multi-drug resistance 1gene (MDR1 gene), genes that allow viral uptake and replication, genesthat cause neurodegenerative disorders, genes that cause proteinaggregation and/or accumulation, genes that cause up-regulation or downregulation of hormones, genes that cause neurological disorders, genesthat cause cardiac disorders, and genes that cause psychologicaldisorders. One of skill in the art would understand which genes areencompassed by the broad categories of exemplary genes described above.

Methods of Manufacture

In certain embodiments, the polymer nanocapsules described herein aremanufactured to achieve a specific size, to target a specific site forgene downregulation, and to downregulate a specific gene. The size ofthe polymer nanocapsules described herein can be determined based on thepolymer:crosslinker ratio as described herein. Targeted delivery can beachieved, for example, using conjugate agents that are attached (i.e.,conjugated) to the exterior of the polymer nanocapsules as describedherein. Furthermore, the specific binding of an RNAi molecule of apolymer nanocapsules described herein to a specific gene (therebydecreasing specific gene expression) can be achieved by designing theRNAi molecule using methods known to one of skill in the art. See, e.g.,Birmingham et al., “A protocol for designing siRNAs with highfunctionality and specificity,” Nature Protocols, 2007; 2(9):2068-78.Furthermore, the ability of the polymer nanocapsules to deliver the RNAimolecules to the target site can be optimized and determined byadjusting the ratios of degradable:nondegradable polymers as describedherein.

Methods of Administration

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal,vaginal and topical (including buccal and sublingual) administration. Inpreferred embodiments, the pharmaceutical compositions are administeredby intravenous or intraparenteral infusion or injection.

EXAMPLES Example 1 Manufacture of Nanocapsules

Highly stabilized siRNA polymer nanocapsules were fabricated through anin situ polymerization technique as described in Example 1 and FIGS. 1and 2. The manufacture of a siRNA nanocapsule platform depicted in FIG.1, starts with a positively charged monomer A, a crosslinker B, aneutral co-monomer C and enriches these molecules around the surface ofthe negatively charged siRNA through electrostatic interaction andhydrogen bonding. A series of positively charged monomers (Table 1),crosslinkers (Table 2) and neutral co-monomers (Table 3) were used inthe manufacture of the polymer nanocapsule platform. Differentcrosslinkers, for example those listed in Table 2, can be used to formcopolymer coatings with tunable composition, structure, surfaceproperty, and functionality.

This in situ polymerization technique introduced a protectivecrosslinked degradable polymer shell covering on the surface of thesiRNA with designed charges and properties. This crosslinked polymershell provides protection to the siRNA from RNase degradation,temperature dissociation and serum inactivation. Non-liming examples ofcrosslinkers that can be used in the siRNA nanocapsules are provided inTable 2.

Example 2 Manufacture of Nanocapsules Using Acryl-Spermine

In this example, the preparation of acryl-spermine was achieved byreacting spermine with acrylic acid, hydroxysuccinimide ester (NAS).Briefly, spermine (100 mg) and NAS (80 mg) were dissolved in 1 mLchloroform, respectively. NAS solution was then added into sperminesolution gradually at room temperature under vigorous stirring. Afterovernight reacting, the mixture was filtered to remove by-products. Thefiltrate was then dried by rotary evaporation, followed by re-dispersingwith ddH2O. After removal of insoluble substance, the solution waslyophilized. Finally, acryl-spermine was purified by process TLC. One ofskill in the art that this is an exemplary method of manufacturingacryl-spermine and will understand that alternative methods could beused to reach the same result.

As illustrated in FIG. 2, the manufacture of a nanocapsules began with apositively charged, polymerizable monomer. For purposed of this example,the positively charged, polymerizable monomer is acryl-spermine whichwas prepared as described above. The first step of the process ofmanufacturing the polymer nanocapsules required electrostaticinteractions enriched around the surface of the negatively chargedsiRNA. After the initial interactions, subsequent room-temperaturepolymerization in an aqueous solution took place with the pH-degradablecrosslinkers and hydrophilic monomers. During the room temperaturepolymerization, each siRNA molecule was wrapped in a thin shell ofpolymer network. Such a crosslinked shell served to protect the coredsiRNA from hydrolysis. Specific tuning of the ration of theacryl-spermine and the hydrophilic monomers allowed precise control ofthe surface charge and ensured the effective cellular transduction ofthe polymer nanocapsules. Moreover, 2 is stable in neutral pH but arerapidly degraded in acidic environment, such as late endosomes withpH˜5.4. We believe that this unique responsive design will provide thepolymer nanocapsules with outstanding stability in serum (pH˜7.4), whileenabling their escape from endosomes upon the degrading of the shellthat releases the siRNA to the cytoplasma (Step IV). Furthermore, oursynthetic approach allows for the immobilization of targeting components(e.g., antibody) to the polymer nanocapsules surface, allowing targetingdelivery of siRNA.

Example 3 Ability of Nanocapsules to Protect and Deliver EncapsulatedsiRNA

In order to test the efficacy of the polymer nanocapsules describedherein, a single siRNA nanocapsule platform that efficiently deliverssiRNA was generated. Schematics of how the polymer nanocapsules are madeare depicted in FIG. 1 and FIG. 2. These nanocapsules of this platformwere designed to protect the encapsulated siRNA from nucleases and caneffectively deliver siRNA into cells.

In order to test the ability of the polymer nanocapsules to protect theencapsulated siRNA and effectively deliver the siRNA into cells, thewell known siRNA1005 was used in the preparation of the polymernanocapsules. siRNA1005 is a short hairpin RNA (shRNA) that targets theHIV-1 co-receptor CCR5. The well-characterized potency of siRNA1005 madeit an ideal candidate for demonstrating CCR5 knockdown by applying thesingle molecule nanocapsule delivery technology. Exemplary images ofnanocapsules with siRNA1005 are presented in FIG. 4 and FIG. 5.

As proof of concept we showed that CCR5 RNA can be effectively knockeddown by nanocapsules of siRNA1005. One unique advantage of thisnanocapsule platform is the ability to modify the polymer nanocapsulesby selecting monomers and crosslinkers which alter the chemicalproperties of size, charge, and reactivity of the particles.Non-limiting examples of monomers and crosslinkers can be found in Table1 and Table 2.

The ability of the polymer nanocapsules to effectively protectencapsulated materials from degradation, was also demonstrated byencapsulating proteins. For example, EGFP, HRP, and caspase 3 wereencapsulated and protected in the polymer nanocapsules. Furthermore, thepolymer nanocapsules were also used to encapsulated quantum dots andsiRNAs such as siEGFP, siLuciferase, and si1005 as described above.

Example 4 Delivery of siRNA Using DNA that Encodes shRNA

Delivery of siRNA was accomplished using DNA that encodes shRNAtranscriptional units. For example, plasmid and viral vectors were usedbecause they provide high levels and long term expression of the siRNA.

In this example, the polymer nanocapsule technology was further extendedto nanocapsules of DNA encoding shRNA1005. As described in Example 3,shRNA1005 is an RNAi that targets and knocks down the expression of CCR5RNA expression. This DNA nanocapsule technology is more challenging thanthe above described RNAi nanocapsule because DNA transcription units aremuch larger than siRNA.

The formulation of polymer nanocapsules platform for encapsulation ofDNA cassettes was modified by tuning monomers and crosslinkers whichalter the size, charge, and degradability of the particles. Non-limitingexamples of monomers and crosslinkers are provided in Table 1 and Table2.

Of particular interest, the polymer nanocapsules were engineered fordelayed release using crosslinkers that degrade at different rates,enabling effective siRNA activity over several days or weeks. This is abig advantage over current small molecule drugs that are required dailyadministration

Example 5 Synthesis and Delivery of DNA Nanocapsules

As illustrated in FIG. 1, starting with the monomer A and B, crosslinkerC, these molecules self-assembled along the surface of the DNA cassettesthrough electrostatic interaction and hydrogen bonding (Step 1). Then athin network of polymer shell was formed around the DNA cassette bysubsequent in situ polymerization (Step 2), which effectively conferednew surface properties that protect the DNA. Crosslinker C was anon-degrable crosslinker. Acid-degradable crosslinkers D were stable inneutral pH but were rapidly degraded in acidic environment, such as lateendosomes with pH around 5.4.

This unique responsive design provided the polymer nanocapsules withoutstanding stability in the physiological pH of serum (pH˜7.4). It alsoled to effective endosomal escape due to the “proton-sponge” effectresulting from the positive charges of monomers B and enabledcontrollable release of the DNA cassette into the cytoplasm upon thedegradation progress of the shell. The DNA cassette then entered thenucleus and allowed the endogenous generation of siRNA (Step 4).

Example 6 DNA Nanocapsule Incorporating Short Linear DNA Cassettes

As proof of concept, we designed a model DNA nanocapsule incorporating ashort linear DNA cassette with H1 expressed sh1005 shRNA and H1expressed anti-EGFP shRNA as control. Delivery of a large DNA plasmid,consisting of shRNA transcriptional units and antibiotic resistancegenes, was hindered by delivering barriers at the cell membrane andnucleopore. Accordingly a second DNA cassette was manufactured using aminimized linear DNA cassette of only 395 base pairs that was producedby PCR. The DNA cassette of only 395 base pairs, was able to transfernucleopores more efficiently.

Gel electrophoresis image of these two DNA cassettes was shown in FIG.3A. Interestingly, the TEM image of linear naked DNA cassette stainedwith tungsten agent appeared as a dark half circular arc with a diameterabout 50 nm. In contrast, the DNA cassette nanocapsules had a roundmorphology with a much smaller size of approximately 30 nm. This islikely a result of DNA condensation through complexing with polymers.

Compared to previously reported nanoparticles that contain plasmid DNA(approximately 150-300 nm), the size of DNA cassette nanoparticles isremarkably 1/10 to ⅕ the size. This small size of the DNA cassettenanocapsules likely leads to a high diffusional rate and improve thedelivery efficiency.

Example 7 DNA Cassette Nanocapsules are Efficiently Delivered to Cells

DNA cassette nanocapsules of this invention can be effectively deliveredto cells. The optical and fluorescent images of HEK-293T cells afterincubation with Alexa592-labeled DNA cassette nanocapsules for 4 hrs isshown in FIG. 4 and FIG. 5. The intense green fluorescence demonstratesdelivery of the FITC-labeled siRNA nanocapsules (FIG. 4 and FIG. 5).

Flow cytometry of HEK-293 T cells transduced with Alexa592-labeled DNAcassette nanocapsules confirmed the results of the fluorescence imagingand demonstrated successful delivery of fluorescence-labeled siRNAnanocapsule (FIG. 5C).

RNAi activity of the DNA nanocapsule in 293T cells expressing a fusionof CCR5 and luciferase reporter gene sequence with CCR5-shRNA (sh1005)was also examined (FIG. 5D). The CCR5-shRNA DNA nanocapsulesdown-regulated about 80% of the bioluminescence intensity reflecting theknockout of the CCR5-luciferase fusion mRNA while cells treated withcontrol DNA cassette nanocapsules did not exhibit significant decreasesin the luciferase activity.

To investigate the sensitivity of DNA nanocapsules against Dnase I, DNAcomplexed with Lipofectamine® and DNA nanocapsules were incubated withDnase I for 1 hour (FIG. 6A).

After acid treatment and DNA extraction, agarose gel electrophoresisshowed such nanocapsules were able to proted and maintain the integrityof the encapsulated DNA. In contrast, non-encapsulated DNA in the nativestate and non-encapsulated DNA that was formulated with Lipofectamine®degraded. The DNA nanocapsules did not show obvious cytotoxicity at theconcentration of DNA cassette below 0.4 pmol. At 0.4 pmol, the viabilityof cells treated with DNA nanocapsules was slightly reduced to about 85%(FIG. 7).

Example 8 siRNA Nanocapsules are Efficiently Delivered to Cells andTarget Specific Sequences

FIG. 4A shows a representative TEM image of the double-stranded siRNAnanocapsules which target CCR5 sequence 3′-gagcatgactgacatctac-5′ withan average diameter of 25 nm. Interestingly, within each nanocapsule, adark core with diameter around 5 nm was clearly observed, which is dueto preferred complexation of siRNA with the tungsten-staining agent usedfor TEM observation. Since a double-stranded siRNA (21 base pairs) hasan average molecular weight of 12 kDa and size of 3-5 nm, each of thepolymer nanocapsules appears to only contain one siRNA molecule. FIG. 4Bshows a fluorescent image of HEK-293 T cells after incubation withFITC-labeled siRNA for 4 hrs. The intense green fluorescence proves theeffective delivery of the siRNA nanocapsules.

As proof of concept, CCR5-siRNA was used to target and down-regulateCCR5 expression. It has been well demonstrated that individuals bornwith naturally existing mutations in the CCR5 chemokine receptor areprotected from HIV infection and disease progression. CCR5-siRNA holdsgreat promise as a therapeutic drug to downregulate CCR5 expression andto develop HIV resistance in patients. To prove this concept, FIG. 4Cshows flourescence images of HEK 293 cells transducted with siRNAnanocapsules targeted to CCR5 sequence (left panel of image in FIG. 4C)and EGFP sequence (right panel of image in FIG. 4C). Clearly, deliveryof the CCR5-siRNA nanocapsules effectively down-regulates theCCR5-mCherry fusion protein expression. This demonstrates theeffectiveness of siRNA nanocapsules delivery and function.

Luciferase-expressing CWR cells stably expressing luciferase were usedto test the gene-silencing efficacy of single siRNA nanocapsules (FIG.14A). Cells treated with luciferase siRNA nanocapsules showed asignificant decrease in the luciferase activity especially atconcentrations above 50 nM, while cells treated with control siRNAnanocapsules did not exhibit significant decrease in the luciferaseactivity. The siRNA nanocapsules did not show obvious cytotoxicity atthe concentration of siRNA below 200 nM. At 300 nM, the viability ofcells treated with siRNA nanocapsules was slightly reduced to about 75%(FIG. 14B).

Furthermore, without human serum, nanocapsules and Lipofectamine®(Invitrogen®) silenced expression of CCR5-mCherry expression to 8% and15%, respectively. But in the presence of human serum, CCR5 siRNAnanocapsules still knocked down more than 85% of CCR5-mCherry expressionwhile siRNA delivered through Lipofectamine® only made a number at 45%.Therefore, nanocapsules can provide extra protection and stabilizationto siRNA inside against attacking of human serum nucleases compared withLipofectamine®.

Example 9 DNA Cassette Nanocapsules Efficiently Knockdown GeneExpression in Cells

The knockdown efficacy of DNA cassette nanocapsule was compared tostandard Lipofectamine® siRNA transduction to HEK 293T cells expressingCCR5-luciferase fusion protein (FIG. 8).

After 48 hours, 0.1 pmol of sh1005 DNA cassette nanocapsule silenced theexpression of CCR5-luciferase to 45%. In stark contrast, 100 pmol (1000×the amount) of siRNA-Lipofectamine® complex was required to knock downthe level of CCR5-luciferase to 47%. On a molar basis, the sh1005 DNAcassette nanocapsule is over 1000-fold more effective at downregulatingCCR5 than si1005 siRNA formulated with Lipofectamine® (FIG. 7 and FIG.8). This result is likely due to de novo transcription of shRNA withintransduced cells.

Example 10 Delayed Release and Degradable Nanocapsules

Because of the high potency of the DNA cassette nanocapsules, DNAcassette nanocapsules can be used in applications where sustainedactivity is beneficial. To accomplish this, DNA cassette nanocapsuleswere engineered for delayed release using crosslinkers that degrade atdifferent rates.

The DNA nanocapsules prepared with 100% degradable crosslinkers (e.g.,Glycerol 1,3-diglycerolate diacrylate, GDGDA) (5:0) was degradedcompletely after 10 hours. The DNA nanocapsules prepared with a mixtureof one part degradable crosslinker to four parts non-degradablecrosslinker (e.g., N,N′-methylene bisacrylamide, BIS) was completelydegraded after 150 hours (FIG. 9). The slopes of the degradationprofiles consistently increase with the percentage of degradablecrosslinkers. This confirms that a higher percentage of the degradablecrosslinkers leads to a higher degradation rate.

Using 100% of degradable crosslinker, the downregulation of CCR5 reached70% at day 3 and 5 following transduction and decreased to 23% at day 9(FIG. 10).

When the ratio of degradable crosslinker to non-degradable crosslinkeris 3:2, the knockdown of CCR5 increased from 58% at day 3 to 70% at day5 and then decreased to 42%.

The DNA nanocapsule with the ratio of degradable crosslinker tonon-degradable crosslinker at 2:3, the silencing percentage of the CCR5increased from 53% to 64% and further increased to 73%. By using 20%degradable crosslinker, the down regulation of CCR5 was as low as 20% atday 3 and reached 80% at day 9.

Example 11 Visualization Nanocapsules

IR spectra of the polymer nanocapsules were obtained on a PerkinElmerParagon 1000 FT-IR spectrometer. UV-Visible spectra were acquired with aGeneSys 6 spectrometer (Thermo Scientific). Fluorescence spectra wereobtained with a QuantaMaster Spectrofluorimeter (Photon TechnologyInternational). TEM images of nanocapsules were obtained on a PhilipsEM120 TEM at 100000× (see, e.g., FIG. 3, FIG. 4, and FIG. 5).

Before observation, siRNA nanocapsules were negatively stained using 1%pH 7.0 phosphotungstic acid (PTA) solution. Zeta potential and particlesize distribution were measured with a Malvern particle sizer Nano-ZS.SEM images of nanocapsules were obtained with a JEOL JSM-6700F SEM. Drysamples on a silicon surface were sputter-coated with gold beforemeasurement. Fluorescent images of cells were obtained with either ZeissAxio Observer.Z1 fluorescence microscope or Leica TCS SP MP InvertedConfocal Microscope. Cellular fluorescent intensity distribution wasdetermined with Becton Dickinson FACScan Analytic Flow Cytometer. A 488nm argon laser was used as the excitation light.

Example 12 Synthesis of siRNA Nanocapsules

In situ polymerization and the process of manufacturing siRNAnanocapsules with different types and ratios of siRNA was optimized.Specifically, positively charged monomer, hydrophilic monomer anddegradable crosslinkers were used to optimize the siRNA nanocapsules.

The effects of buffer salt, ion types, ionic strength and solventcomposition on the morphology and yield of siRNA nanocapsules wasassessed.

Varieties of targeting components were conjugated to the siRNAnanocapsules to achieve targeted delivery of the siRNA. For example,targeting conjugates used in these experiments included CD4, CD8, CD45,aHLA, and transferrin. These exemplary conjugates can be used alone orin combination to achieve specific targeting of the polymernanocapsules.

FIG. 22 depicts the targeting delivery of siRNA to 293 cells expressingCD4 receptors. FIG. 22A shows the flow cytometry graph of 293 cellsexpressing CD4 receptors after incubation with FITC-labeled siRNAnanocapsules conjugated with anti-CD4 antibody for 4 hours. FIG. 22Bshows the flow cytometry graph of regular 293 cells without CD4receptors after incubation with FITC-labeled siRNA nanocapsulesconjugated with anti-CD4 antibody for 4 hours.

Example 13 Characterization of the Nanocapsules

TEM and dynamic light scattering were used to determine the size andsize distribution of single-siRNA nanocapsules. Furthermore,electrophoresis and electrophoretic light scattering was used toinvestigate the surface charge and the interaction between siRNA andnanocapsules. Specifically, size, surface charge, and encapsulationyield were investigated.

The stability of siRNA and single-siRNA nanocapsules in the presence ofnuclease and serum was compared. Degradability and releasing profile ofsiRNA nanocapsules has been investigated in the buffer of pH 7.4 and5.4. siRNA complexed with lipofectamine and siRNA nanocapsules wasincubated with nuclease and human serum for 1 hour. After RNAextraction, agarose gel electrophoresis showed such nanocapsules couldmaintain the integrity of siRNA inside (FIG. 6B), while siRNA isdegraded at the same time in the native state or when formulated withlipofectamine.

Example 14 Intracellular Delivery of the siRNA Nanocapsules

The siRNA delivery efficiency of the siRNA nanocapsules was tested in abroad variety of cells. Examples of these cells include HEK-293 T, Hela,CEM, PBMCs, and MSCs.

Fluorescence-labeled siRNA was used to investigate the endocytosispathway using endocytosis inhibitors. The efficiency and toxicity ofsiRNA delivery by nanocapsules was compared with those by the commercialliposome agents. Different types of siRNA including, CCR5, EGFP, Gaussialuciferase was used to quantitatively assess specificity of genesilencing.

Example 15 In Vitro Cellular Internalization

Cellular internalization studies were performed via fluorescencemicroscopic technique and fluorescence-activated cell sorting (FACS).HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% bovine growth serum (BGS) and 1%penicillin/streptomycin. Cells (20000 cells/well, 24-well plate) wereseeded the day before adding the siRNA nanocapsules.

siRNA nanocapsules with different concentrations were added into thecell medium. After incubation at 37° C. for 2 to 4 hrs, the cells werewashed three times with PBS and either visualized with a fluorescentmicroscope or trypsinized, centrifuged, and re-suspended in PBS andanalyzed via FACS.

Example 16 Cell Proliferation Assay

The toxicity of the polymer nanocapsules was assessed by the MTT assayusing native proteins as control. HEK 293T cells (7000 cells/well) wereseeded on a 96-well plate the day before exposure to the polymernanocapsules. Nanocapsules with different concentrations were incubatedwith the cells for 2-4 hrs, removed from the mixture, and incubated withfresh media for 24 hrs. The MTT solution (20 μL) was added to each welland incubated for 3 h. The medium was then removed and 100 μL DMSO wasadded onto the cells. The plate was placed on a shaking table, 150 rpmfor 5 min to thoroughly mix the solution, and then absorbance readingswere measured at 560 nm. Untreated cells were used as the 100% cellproliferation control.

Example 17 Synthesis of Positively Charged Monomers for siRNANanocapsules

The preparation of N-(3-((4-aminobutyl)amino)propyl)acrylamide,N-(2-((2-aminoethyl)(methyl)amino)ethyl)acrylamide,N-(piperazin-1-ylmethyl) acrylamide, andN-(2-(bis(2-aminoethyl)amino)ethyl)acrylamide (i.e., positively chargedmonomers) was achieved by reacting amine-containing precursor(N-(3-aminopropyl)butane-1,4-diamine/N-methylpropane-1,3-diamine/piperazin-1-ylmethanamine/N,N′-bis(2-aminoethyl)ethane-1,2-diamine)with acrylic acid, hydroxysuccinimide ester (AHS). Briefly,amine-containing precursors and AHS were dissolved in chloroform at 0.5mol/L, respectively. Then, acrylic acid, hydroxysuccinimide ester wasadded into each of the amine-containing precursors at the molar ratio of1:1 gradually at room temperature under vigorous stirring. Afterovernight reaction, the mixture was filtered to remove by-products. Thefiltrate was then dried by rotary evaporation, followed by re-dispersingin water. After removal of insoluble substance, the solution waslyophilized. Finally, the product was purified by thin layerchromatography. The yield was from 37% to 63%. ^(1H)NMR was performed toconfirm the final products.

^(1H) NMR for N-(3-((4-aminobutyl)amino)propyl)acrylamide produced thefollowing peaks at 400 MHz, D₂O: 6.46 (m, 1H, CH₂═CHCO), 5.65 (m, 2H,CH₂═CHCO), 3.27 (m, 2H, CONH—CH₂), 2.78 (m, 10H, CH₂—NH—CH₂ andCH₂—NH₂), 1.75 (m, 4H, NH—CH₂—CH₂), 1.23 (m, 4H, NH—CH₂—(CH₂)₂—CH₂—NH)

^(1H) NMR for N-(piperazin-1-ylmethyl) acrylamide produced the followingpeaks at 400 MHz, D₂O: 6.53 (m, 1H, CH₂═CHCO), 5.69 (m, 2H, CH₂═CHCO),3.91 (m, 2H, CONH—CH₂—N), 2.28 (m, 4H, CH₂—NH—CH₂), 2.75 (m, 4H,CH₂—NH—CH₂).

^(1H) NMR for N-(2-((2-aminoethyl)(methyl)amino)ethyl)acrylamide (400MHz, D₂O): 6.39 (m, 1H, CH₂═CHCO), 5.58 (m, 2H, CH₂═CHCO), 3.18 (m, 2H,CONH—CH₂—N), 2.54 (m, 2H, CH₂—NH—CH₃), 3.22 (m, 3H, NH—CH₃)

^(1H) NMR for N-(2-(bis(2-aminoethyl)amino)ethyl)acrylamide produced thefollowing peaks at 400 MHz, D₂O: 6.41 (m, 1H, CH₂═CHCO), 5.62 (m, 2H,CH₂═CHCO), 3.14 (m, 2H, CONH—CH₂—N), 2.48 (m, 6H, N—(CH₂)₃), 2.65 (m,4H, CH₂—NH₂).

Example 18 Synthesis of Positively Charged Monomers for siRNANanocapsules

The preparation ofN-(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)methacrylamide,N-(3-((4-aminobutyl)amino)propyl)methacrylamide,N-(2-((2-aminoethyl)(methyl)amino)ethyl)methacrylamide,N-(piperazin-1-ylmethyl) methacrylamide, andN-(2-(bis(2-aminoethyl)amino)ethyl)methacrylamide (i.e., positivelycharged monomers) was achieved by reacting(N,N′-(butane-1,4-diyl)bis(propane-1,3-diamine)/N-(3-aminopropyl)butane-1,4-diamine/N-methylpropane-1,3-diamine/piperazin-1-ylmethanamine/N,N-bis(2-aminoethyl)ethane-1,2-diamine))with methacrylic acid, hydroxysuccinimide ester. Briefly,amine-containing precursors and methacrylic acid, hydroxysuccinimideester were dissolved in 1 mL chloroform at 0.5 mol/L, respectively. TheMAHS was then added into one of the amine-containing precursor at themolar ratio of 1:1 gradually at room temperature under vigorousstirring. After overnight reacting, the mixture was filtered to removeby-products. The filtrate was then dried by rotary evaporation, followedby re-dispersing with ddH2O. After removal of insoluble substance, thesolution was lyophilized. Finally, the product was purified by thinlayer chromatography. The yield was from 32% to 61%. ^(1H)NMR wasperformed to confirm the final products.

^(1H) NMR for N-(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)methacrylamide produced the following peaks at 400 MHz, D₂O: 1.89 (m,3H, CH₂═C(CH₃)CO), 5.72 (m, 2H, CH₂═C(CH₃)CO), 3.27 (m, 2H, CONH—CH₂),2.78 (m, 10H, CH₂—NH—CH₂ and CH₂—NH₂), 1.75 (m, 4H, NH—CH₂—CH₂), 1.23(m, 4H, NH—CH₂—(CH₂)₂—CH₂—NH).

^(1H) NMR for N-(3-((4-aminobutyl)amino)propyl)methacrylamide producedthe following peaks at 400 MHz, D₂O: 1.92 (m, 3H, CH₂═C(CH₃)CO), 5.75(m, 2H, CH₂═C(CH₃)CO), 3.27 (m, 2H, CONH—CH₂), 2.78 (m, 10H, CH₂—NH—CH₂and CH₂—NH₂), 1.75 (m, 4H, NH—CH₂—CH₂), 1.23 (m, 4H,NH—CH₂—(CH₂)₂—CH₂—NH).

^(1H) NMR for N-(piperazin-1-ylmethyl) methacrylamide produced thefollowing peaks at 400 MHz, D₂O: 1.90 (m, 3H, CH₂═C(CH₃)CO), 5.72 (m,2H, CH₂═C(CH₃)CO), 3.91 (m, 2H, CONH—CH₂—N), 2.28 (m, 4H, CH₂—NH—CH₂),2.75 (m, 4H, CH₂—NH—CH₂).

^(1H) NMR for N-(2-((2-aminoethyl)(methyl)amino)ethyl)methacrylamideproduced the following peaks at 400 MHz, D₂O: 1.94 (m, 3H,CH₂═C(CH₃)CO), 5.62 (m, 2H, CH₂═C(CH₃)CO), 3.18 (m, 2H, CONH—CH₂—N),2.54 (m, 2H, CH₂—NH—CH₃), 3.22 (m, 3H, NH—CH₃).

^(1H) NMR for N-(2-(bis(2-aminoethyl)amino)ethyl)methacrylamide producedthe following peaks at 400 MHz, D₂O: 1.93 (m, 3H, CH₂═C(CH₃)CO), 5.67(m, 2H, CH₂═C(CH₃)CO), 3.14 (m, 2H, CONH—CH₂—N), 2.48 (m, 6H, N—(CH₂)₃),2.65 (m, 4H, CH₂—NH2).

Example 19 Synthesis of siRNA Nanocapsules

siRNA was dissolved in 20 uL RNase-free water at 20 uM. Then a specificamount of positively charged monomers, tris-acrylamide and glyceroldimethacrylate (molar ratio=5:5:1) dissolved in 0.5 mL deoxygenated anddeionized water was added to the microcentrifugetube. Radicalpolymerization from the surface of the acryloylated protein wasinitiated by adding 0.02 mg of ammonium persulfate dissolved in 2 μL ofdeoxygenated and deionized water and 0.4 μL ofN,N,N′,N′-tetramethylethylenediamine. The reaction was allowed toproceed for 60 min in a nitrogen atmosphere.

Example 20 Effect of Positively Charged Monomers on siRNA Knockdown ofGene Expression

To test the effect of the positively charged monomers provided in Table1, siRNA nanocapsules were prepared with siRNA against luciferase geneexpression with each of the 14 individual positively charged monomers inTable 1. These knockdown experiments were conducted in luciferaseexpressing CWR cells.

To prepare the polymer nanocapsules, siRNA was dissolved in 20 uLRnase-free water at 20 uM. Then a specific amount of a positivelycharged monomer selected from Table 1, tris-acrylamide and glyceroldimethacrylate (total number of protonable amines of positively chargedmonomer:tris-acrylamide:glycerol demethacrylate=15:5:1) were dissolvedin 0.5 mL deoxygenated and deionized water was added to themicrocentrifugetube. Radical polymerization from the surface of theacryloylated protein was initiated by adding 0.02 mg of ammoniumpersulfate dissolved in 2 μL of deoxygenated and deionized water and 0.4μL of N,N,N′,N′-tetramethylethylenediamine. The reaction was allowed toproceed for 60 min in a nitrogen atmosphere.

CWR cells were treated with siRNA nanocapsules at 50 nM for 4 h at 37°C. in serum-free medium. Then mediums were changed to DMEM with 10%Bovine Fetal Serum. After 48 h, the luciferase activity was determinedusing a 96-wells plate reader (FIG. 11).

Example 21 Effect of Different Crosslinkers on siRNA Knockdown of GeneExpression

To test the effect of the crosslinkers provided in Table 2 on geneknockdown, siRNA nanocapsules were prepared with siRNA againstluciferase gene expression with 1,3-glycerol dimethacrylate, Glycerol1,3-diglycerolate diacrylate, N,N′-bis(acryloyl)cystamine,bis[2-(methacryloyloxy)ethyl]phosphate, or N,N′-Methylenebisacrylamide(Table 2). These knockdown experiments were conducted in luciferaseexpressing CWR cells.

To prepare the polymer nanocapsules, siRNA was dissolved in 20 uLRnase-free water at 20 uM. Then a specific amount of acryl-spermine,tris-acrylamide and a crosslinker from Table 2 (total number ofprotonable amines of positively chargedmonomer:tris-acrylamide:crosslinker=15:5:1) were dissolved in 0.5 mLdeoxygenated and deionized water was added to the microcentrifugetube.Radical polymerization from the surface of the acryloylated protein wasinitiated by adding 0.02 mg of ammonium persulfate dissolved in 2 μL ofdeoxygenated and deionized water and 0.4 μL ofN,N,N′,N′-tetramethylethylenediamine. The reaction was allowed toproceed for 60 min in a nitrogen atmosphere.

CWR cells were treated with siRNA nanocapsules at 50 nM for 4 h at 37°C. in serum-free medium. Then mediums were changed to DMEM with 10%Bovine Fetal Serum. After 48 h, the luciferase activity was determinedusing a 96-wells plate reader (FIG. 12).

Example 21 Effect of Different Neutral Monomers on siRNA NanocapsuleSize

To test the effect of the neutral monomers provided in Table 3 onnanocapsules size, siRNA nanocapsules were prepared with1N-(1,3-dihydroxy-2-(hydroxymethyl) propan-2-yl) acrylamide, acrylamide,N-(hydroxymethyl)acrylamide, 2-hydroxyethyl acrylate, or 2-hydroxyethylmethacrylate (Table 3).

To prepare the polymer nanocapsules, siRNA was dissolved in 20 uLRnase-free water at 20 uM. Then a specific amount of acryl-spermine, aneutral monomer selected from Table 3, and glycerol dimethacrylate(total number of protonable amines of acryl-spermine:neutralco-monomer:glycerol demthacrylate=15:5:1) were dissolved in 0.5 mLdeoxygenated and deionized water was added to the microcentrifugetube.Radical polymerization from the surface of the acryloylated protein wasinitiated by adding 0.02 mg of ammonium persulfate dissolved in 2 μL ofdeoxygenated and deionized water and 0.4 μL ofN,N,N′,N′-tetramethylethylenediamine. Once the siRNA nanocapsules wereformed, the size of the polymer nanocapsules were measured (FIG. 13).

1. A polymer nanocapsule comprising a polymer shell and a RNAi molecule,wherein the polymer shell comprises a) one or more positively chargedmonomers selected from Table 1, b) one or more crosslinkers selectedfrom Table 2, and c) one or more neutral monomers selected from Table 3;and wherein the RNAi molecule is siRNA or an shRNA DNA cassette.
 2. Thepolymer nanocapsules of claim 1, wherein the one or more crosslinkerscomprise a ratio of degradable crosslinker to non-degradablecrosslinker.
 3. The polymer nanocapsules of claim 1 or 2, wherein ratioof degradable crosslinker to non-degradable crosslinker is selected fromthe ratios comprising 1:0, 3:2, 2:3, or 1:4.
 4. The polymer nanocapsulesof claim 1, wherein all of the crosslinkers are degradable crosslinkers.5. The polymer nanocapsules of claim 4, wherein all the degradablecrosslinkers are glycerol 1,3-diglycerolate diacrylate.
 6. The polymernanocapsules of any of claims 1-5, wherein the one or more positivelycharged monomers is acryl-spermine.
 7. The polymer nanocapsules of anyof claims 1-5, wherein the one or more positively charged monomers isselected from the group comprising N-(3-Aminopropyl) methacrylamidehydrochloride, Dimethylamino ethyl methacrylate,(3-Acrylamidopropyl)trimethylammonium hydrochloride, and(3-Acrylamidopropyl)trimethylammonium hydrochloride.
 8. The polymernanocapsules of any of claims 1-5, wherein the one or more positivelycharged monomers is selected from the group comprisingN-(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)methacrylamide,N-(3-((4-aminobutyl)amino)propyl)acrylamide,N-(3-((4-aminobutyl)amino)propyl)methacrylamide,N-(2-((2-aminoethyl)(methyl)amino)ethyl)acrylamide,N-(2-((2-aminoethyl)(methyl)amino)ethyl) methacrylamide,N-(piperazin-1-ylmethyl)acrylamide,N-(piperazin-1-ylmethyl)methacrylamide,N-(2-(bis(2-aminoethyl)amino)ethyl)acrylamide, andN-(2-(bis(2-minoethyl)amino)ethyl)methacrylamide.
 9. A polymernanocapsule of claim 1, wherein the polymer nanocapsules comprisesacryl-spermine, tris-acrylamide, and crosslinker selected from Table 2.10. The polymer nanocapsules of any of claims 1-8, wherein the one ormore positively charged monomers has 3 protonable amines.
 11. Thepolymer nanocapsules of any of claims 1-8, wherein the one or morepositively charged monomers has 2 protonable amines.
 12. The polymernanocapsules of any of claims 1-8, wherein the one or more positivelycharged monomers has 1 protonable amines.
 13. The polymer nanocapsulesof any of claims 1-12, wherein the polymer nanocapsules areapproximately 20 nm to 250 nm in diameter.
 14. The polymer nanocapsulesof any of claims 1-13, wherein the polymer nanocapsules is conjugated toa targeting agent.
 15. The polymer nanocapsules of claim 14, wherein thetargeting agent is selected from the group comprising cyclodextrin,adamantine, CD4, CD8, CD45, aHLA, and transferrin.
 16. The polymernanocapsules of claim 14 or 15, wherein the targeting agent delivers thepolymer nanocapsules to a specific cell type, wherein the cell type isselected from the group comprising immune cells, blood cells, cardiaccells, lung cells, optic cells, liver cells, kidney cells, brain cells,cells of the central nervous system, cells of the peripheral nervoussystem, cancer cells, cells infected with viruses, stem cells, skincells, intestinal cells, and/or auditory cells.
 17. The polymernanocapsules of claim 16, wherein the cancer cells are cells selectedfrom the group comprising lymphoma cells, solid tumor cells, leukemiacells, bladder cancer cells, breast cancer cells, colon cancer cells,rectal cancer cells, endometrial cancer cells, kidney cancer cells, lungcancer cells, melanoma cells, pancreatic cancer cells, prostate cancercells, and thyroid cancer cells.
 18. The polymer nanocapsules of any ofclaims 1-17 further comprising a pharmaceutically acceptable carrier.19. A pharmaceutical composition comprising one or more polymernanocapsule of any of claims 1-18.
 20. A method of treating a diseasecharacterized by over expression of a gene with a pharmaceuticalcomposition of claim 19, wherein the siRNA or shRNA knocks down ordecreases expression of an over expressed gene, thereby treating thedisease.
 21. A polymer nanocapsule comprising a polymer shell and a RNAimolecule, wherein the polymer shell comprises a) one or more positivelycharged monomers, b) one or more crosslinkers, and c) one or moreneutral monomers; and wherein the RNAi molecule is siRNA or an shRNA DNAcassette; wherein the one or more positively charged monomers isselected from the group comprising:

wherein R²⁰ is unsubstituted C₁-C₆ alkyl R²¹ is selected from the groupconsisting of:

wherein m is an integer from 1 to 5; R²² is H or unsubstituted C₁-C₆alkyl or C₁-C₆ alkyl substituted with NR²⁵R²⁶, wherein R²⁵ and R²⁶ areindependently selected from H or unsubstituted C₁-C₆ alkyl or C₁-C₆alkyl substituted with amino or C₁-C₆ alkyl substituted with NR²⁷R²⁸,wherein R²⁷ and R²⁸ are independently selected from H or unsubstitutedC₁-C₆ alkyl or C₁-C₆ alkyl substituted with amino R²³ is H orunsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substituted with amino or C₁-C₆alkyl substituted with NR²⁹R³⁰, wherein R²⁹ and R³⁰ are independentlyselected from H or unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substitutedwith amino or C₁-C₆ alkyl substituted with NR³¹R³², wherein R³¹ and R³²are independently selected from H or unsubstituted C₁-C₆ alkyl or C₁-C₆alkyl substituted with amino and R²² and R²³ are optionally combined toform a 5-7 membered heterocycloalkyl ring; and R²⁴ is a lone pair ofelectrons or unsubstituted C₁-C₆ alkyl. wherein the one or morecrosslinker is selected from the group comprising:

wherein R¹ is unsubstituted C₁-C₆ alkyl, R⁴ is unsubstituted C₁-C₆alkyl, and A is selected from the group consisting of:

wherein R¹⁰ is unsubstituted C₁-C₆ alkylene; an amino acid; a peptideconsisting of from 2 to 10 amino acids;

wherein R¹¹ is unsubstituted C₁-C₆ alkylene and R¹² is unsubstitutedC₁-C₆ alkylene;

wherein n is from 1 to 10; and

wherein R¹³ is unsubstituted C₁-C₆ alkylene and R¹⁴ is unsubstitutedC₁-C₆ alkylene; and wherein the one or more neutral monomers is selectedfrom the group comprising:

wherein R1 is unsubstituted C1-C4 alkyl and R2 is amino or aminosubstituted with hydroxy substituted alkyl or OR³, wherein R3 is hydroxyalkyl.
 22. The polymer nanocapsules of any of claims 1 to 21, whereinthe polymer nanocapsules comprises a single RNAi molecule.
 23. A methodof making a siRNA polymer nanocapsule, the method comprising a)dissolving the siRNA in RNase-free water, b) dissolving one or morepositively charged monomers selected from Table 1 and one or morecrosslinkers selected from Table 2 in deoxygenated and deionized waterto create a monomer mixture, c) combining the dissolved siRNA of Step(a) with the monomer mixture of Step (b), d) adding ammonium persulfateand N,N,N′,N′-tetramethylethylenediamine to the product of Step (c), ande) incubating the product of Step (d) in serum-free medium.